Complete nutrient medium for use as a cosmetic and cosmetic use thereof

ABSTRACT

A cosmetic composition contains an aqueous complex nutritive base comprising a plurality of amino acids, at least one vitamin, a plurality of assimilable organic components, and at least one inorganic salt. The cosmetic composition does not contain a biological extract of animal or cellular origin, or a living nourishing substrate. A cosmetic method comprises contacting human skin with the cosmetic composition.

[0001] The present invention relates to a complex nutrient medium, toits applications and more especially to its use for manufacturing acomposition for topical use, and in particular for topical cosmetic ormedicinal use.

[0002] The composition obtained according to the invention enables anextracellular environment which is entirely suited to the epidermis tobe obtained, by supplying in particular:

[0003] an optimized nutritional provision, both in respect of vitaminsand trace elements and in respect of essential amino acids,

[0004] cell growth factors directed towards replacing the morphogeniccellular interactions,

[0005] and pH and osmolarity characteristics close to physiologicalconditions.

[0006] Generally speaking, according to the invention, the nutritionalagent consists of a complex nutrient medium comprising compounds whichare both biocompatible, biomimetic and bioavailable in respect of theskin, excluding any biological extract of animal origin, such as foetalcalf serum, or of cellular origin.

[0007] The complex nutrient medium adopted according to the inventionhas a composition suitable for permitting, on its own and in an aqueousmedium, viable in vitro culture of an inoculum of human epidermalkeratinocytes, with at least one clonal proliferation of the latter atthe first passage, without a living nourishing substrate such asfibroblasts.

[0008] “Biocompatible” is understood to mean the property according towhich the compound is harmless to the skin.

[0009] “Biomimetic” is understood to mean the fact that the compound ispresent in the natural state in the skin.

[0010] “Bioavailable” is understood to mean the property according towhich the compound is assimilable by human epidermal keratinocytes, bothin vitro and in vivo.

[0011] By routine tests, a person skilled in the art is in a position toformulate a complex nutrient medium according to the invention, inparticular by carrying out with the said medium in vitro culturing ofkeratinocytes, the growth of which can be observed, for example under amicroscope.

[0012] In this connection, the following documents have alreadydescribed media suited to in vitro culturing of keratinocytes, theviability and growth of which can be determined by the tests currentlyin use, and be directly assessed by observation under a microscope:

[0013] Boyce S T, Ham R G, Calcium-regulated differentiation of normalhuman epidermal keratinocytes in defined clonal culture and serum-freeserial culture, J. Invest. Dermatol. 1983; 81: 33S-40S

[0014] Boyce S T, Ham R G, Cultivation, frozen storage, and clonalgrowth of normal human epidermal keratinocytesin [sic] serum-free media,J. Tissue Culture Methods. 1985; 9: 83-93.

[0015] Where necessary, the content of these publications isincorporated in the present description.

[0016] The complex nutrient medium according to the invention comprisesamino acids, one or more vitamins, one or more cell growth factors andone or more inorganic salts.

[0017] A composition of the invention for topical use comprises a phasewhich is biocompatible with the superficial parts of the human body, inwhich phase at least the said nutrient medium as defined above isdistributed homogeneously.

[0018] In a composition according to the present invention, thebiocompatible phase in which the nutritional agent is distributed canconstitute the excipient, or one of the components of the excipient, ofthe said composition.

[0019] Since all of the compounds present in the nutrient mediumaccording to the invention are water-soluble, two methods of formulationmay be employed in order to obtain a composition for topical use:

[0020] 1) Aqueous continuous phase, containing the nutrient mediumaccording to the invention:

[0021] in the form of an aqueous gel, with the aid of a nonionicwater-soluble polymer of the polysaccharide or cellulose ether type(polymers compatible with the high ionic strength of the medium);

[0022] in the form of an emulsified system (oil-in-water emulsionemploying surfactants that withstand high ionic strengths);

[0023] in the form of a cosmetic serum.

[0024] 2) Oily continuous phase, the discontinuous phase containing thenutrient medium according to the invention:

[0025] in emulsified form, on the understanding that the ionic strengthof the discontinuous phase entails instability of the emulsion; it is,however, possible to formulate lamellar or cylindrical phases havingbetter stability, or alternatively a two-phase system re-emulsifiedimmediately before use by simple shaking;

[0026] by encapsulation:

[0027] in a rigid capsule of the polysaccharide type, dispersed in thelipid phase,

[0028] in a soft capsule of the gelatin type, dispersed in thediscontinuous phase.

[0029] The use of liposomes as an encapsulation delivery agent can beenvisaged in the form of a liposomal gel in an aqueous continuous phase.

[0030] A composition according to the invention can serve as a cosmeticbase. Its nutritional provision is considerably advantageous forimprovement of the viability, maintenance of the integrity and thebalance of the superficial cells of the skin. In particular, it enablesthe primary intrinsic qualities of the skin to be preserved on along-lasting basis, its resistance to damage to be increased and, whereappropriate, its return to a state of balance to be promoted.

[0031] Another subject of the invention is a cosmetic preparationcomprising a base defined above, in which the complex nutrient mediumconstitutes either an active principle, or an excipient in the presenceof other active principles which it is capable of potentiating.

[0032] The complex nutrient medium of the invention can also be used forthe preparation or production of a medicament.

[0033] The use of such a medium on a weakened skin (irritated ordehydrated skins, older skins, etc) enables the skin to return to asatisfactory state, in terms both of trophicity and of hydration of thesuperficial layers of the epidermis.

[0034] A medicinal composition comprising a complex nutrient mediumaccording to the invention can serve as a pharmaceutical formulationbase, in particular a nutrient pharmaceutical formulation base.

[0035] It possesses, in addition, pharmacological properties which willbe demonstrated in the examples. According to an advantageousapplication of a medicinal composition of the invention, it is intendedfor the preservative treatment of grafts after they are removed. It willpreferably take the form of a sterile solution which is especiallysuitable for the cleaning and maintenance of grafts in third-degreeburns victims.

[0036] In addition, a composition as defined above has efficaciousproperties for preventing or treating disorders of cicatrization such asbedsores, varicose ulcers, stretch marks and keloids, and/or a delay ofcicatrization.

[0037] More generally speaking, a composition according to the inventioncan be incorporated in any preparation for use in a pharmaceuticalformulation, as an active principle optionally with other activeprinciples, but also as an excipient as a result of its capacity topotentiate the action of specific active principles.

[0038] The characteristics, applications and advantages of the presentinvention are described in greater detail in Examples 1 to 4 and FIGS. 1to 4 below.

[0039] Example 1 gives an example of a formulation of a composition ofthe invention.

[0040] Example 2 demonstrates the properties of a composition of theinvention compared to known media, in support of the attached drawing inwhich:

[0041]FIG. 1 is a sectional view of human epidermis after 36 hours ofculture in a standard commercial medium designated MCDB 153, marketed,in particular, by IRVINE SCIENTIFIC and GIBCO-BRL,

[0042]FIG. 2 is a sectional view of human epidermis after 36 hours ofculture in a buffered saline solution (PBS), a balanced saline solutioncommonly used in cell culture, and

[0043]FIG. 3 is a sectional view of human epidermis cultured in thenutrient medium of the invention, described in Example 1, at differentculture times:

[0044] A: after 12 hours

[0045] B: after 24 hours

[0046] C: after 36 hours

[0047] Example 3 demonstrates the absence of stimulation of theproliferation of transformed cells by a composition of the inventioncompared to a standard composition, in support of FIG. 4 which depicts adiagram showing the multiplication of transformed cells cultured on amedium of the invention and a standard medium.

[0048] Example 4 illustrates the pharmacological properties of acomposition of the invention: a) on the treatment of grafts; b) oncicatrization.

EXAMPLE 1

[0049] Formulation of a Composition of the Invention TABLE 1Concentration COMPONENTS in mg/l. Amino acids L-Alanine 9.2 L-ArginineHCL [sic] 421.4 L-Asparagine (anhydrous) 14.2 L-Aspartic acid 4.0L-Cysteine HCl.H₂O 42.0 L-Glutamic acid 14.8 L-Glutamine 1754.4 Glycine7.6 L-Histidine HCl.H₂O 50.0 L-Isoleucine 6.0 L-Leucine 131.2 L-LysineHCl 54.0 L-Methionine 13.5 L-Phenylalanine 10.0 L-Proline 34.6 L-Serine126.1 L-Threonine 24.0 L-Tryptophan 9.3 L-Tyrosine 2 Na 2H₂O 11.7L-Valine 70.3 Vitamins and cell growth factors d-Biotin 0.02 Folic acid0.80 Nicotinamide 0.04 Ca D-Pantothenate 0.30 Pyridoxine HCl 0.06Riboflavin 0.04 Thiamine HCl 0.30 Vitamin B₁₂ 0.41 i-Inositol 18.0Putrescine 2 HCl 0.20 Sodium pyruvate 55.0 Thymidine 0.73 Adenine (HCl)24.0 DL-Lipoic acid 0.20 Inorganic components Sodium chloride 6800.0 KCl112.0 Na₂ HPO₄ 284.0 CuSO₄.5H₂0 0.003 Sodium acetate 300.0 (anhydrous)D-Glucose 1080.0 HEPES (piperazine) 6600.0 Phosphorylethanolamine0.06768 Ethanolamine 0.04684 Sodium sulphate 3.4 Sodium bicarbonate1160.0 FeSO₄.7H₂O 1.39 MgCl₂.6H₂O 120.0 CaCl₂.2H₂O from 13.0 to 22.05ZnSO₄.7H₂O 0.144 (NH4)₆ MO₇O₂₄.4H₂O 0.00120 Na₂SiO₃.5H₂O 0.142MnCl₂.4H₂O 0.00002 SnCl₂.2H₂O 0.00011 NH₄ VO₃ 0.00057

EXAMPLE 2

[0050] The cytocompatibility and the performance features of the complexnutrient medium described in Example 1 were tested on cultures of humankeratinocytes in a monolayer, and on human epidermis reconstituted invitro.

[0051] The nutrient medium according to Example 1 permits the culture ofkeratinocytes in a monolayer under optimal conditions of viability forat least 36 hours without the slightest cytotoxic effect manifestingitself.

[0052] In contrast, a traditional survival solution such as PBS(phosphate buffered saline, a balanced saline solution commonly used incell culture) proves cytotoxic from 12 hours of incubation onwards.

[0053] In agreement with FIG. 3, the nutrient medium according to theexample permits culture of normal human epidermis reconstituted underoptimal conditions of viability, without cytotoxic manifestations evenafter 36 hours (FIG. 3C) of contact. The cultures displayed basal,prickle, mast and intact, orthokeratotic cornified cell layers, ofregular and normal stratification.

[0054] On comparing FIG. 3C with FIG. 1, the latter illustrating the useof a standard commercial medium (MCDB 153, marketed, in particular, byIRVINE SCIENTIFIC), it is seen that the performance features of themedium of the invention are equally good.

[0055] In contrast, the use of PBS induces, in agreement with FIG. 2,the appearance of keratinocytes in a terminal phase of differentiationat the level of the basal and prickle strata, with more or lesspronounced signs of necrosis. A total detachment of the epidermis isalso noted, with complete loss of structuring of the differentkeratinocytic strata.

EXAMPLE 3 Effects of a Composition of the Invention on the Growth ofTransformed Epidermal Cells

[0056] The composition used for this study is the one described inExample 1, comprising the medium termed medium 1.

[0057] The effect of the composition 1 on the growth of a spontaneouslytransformed line of human keratinocytes was tested over 4 days ofculture by comparison with cells cultured on a standard medium (DMEM,Dulbeco Modified Epidermal Medium+foetal calf serum).

[0058] The cells are first inoculated into the standard medium and growuntil the 2nd day after inoculation into this medium. On the 2nd day,the batch of cells is divided into two, one batch continuing to becultured in standard medium, the other in medium 1.

[0059] The results are collated in FIG. 4, in which the curve obtainedwith the points -□- corresponds to the composition of the invention andthat obtained with the points --□-- corresponds to the composition ofstandard medium. The points were duplicated and the counts originatefrom quadruplicates. The results are corrected for the standard error ofthe mean, SEM. The arrow seen in the diagram corresponds to the dividingof the batch on the second day of culture.

[0060] The morphology of the cells differs according to the mediumemployed. That of the cells cultured in medium 1 resembles more closelythat obtained using a semi-defined medium for epithelial cells, of theGIBCO-BRL KSFM type (cells with looser junctions, less pave-mentalappearance, etc).

[0061] No significant difference is noted in the growth of this line inaccordance with the different media, up to confluence (days 6 to 7, notshown here).

[0062] It is concluded that the composition 1 has no stimulatory effecton the proliferation of transformed keratinocytes.

EXAMPLE 4 Effects of a Composition of the Invention on the Taking ofHuman Skin Grafts and the Prevention of Cicatrization Disorders

[0063] The composition tested is the one described in Example 1,comprising the medium termed medium 1.

[0064] The effects of the composition 1 on the taking of human skingrafts and the prevention of cicatrization disorders were studied on amouse model (athymic mouse lacking cell-mediated immunity).

[0065] Two types of grafts were employed: cultured epidermis and humanskins originating from plastic surgery. The grafts were irrigated for 30days with 1 ml of composition 1 (one application daily) for the group Amice and 1 ml of buffered saline solution (PBS) for the group B mice (20animals per group). Compresses of tulle gras were applied after eachirrigation in order to prevent the grafts from drying out.

[0066] A clinical observation of the grafts was carried out on D-7, D-15and D-30.

[0067] Two parameters were evaluated: the necrosis of the culturedepidermis and the cicatrization.

[0068] a) The Necrosis of the Cultured Epidermis (“Taking of Grafts”)

[0069] Scoring is performed from 0 to 3: 0=no sign of necrosis; 1=slightinflammation and superficial degradation of the graft; 2=partialnecrosis; 3=total necrosis.

[0070] The results are collated in Table 2. TABLE 2 Score D-7 D-15 D-30GROUP A MICE (20 grafts in total, treated with the nutrient composition)0 9/20 12/20  16/20  1 7/20 4/20 0/20 2 3/20 2/20 2/20 3 1/20 2/20 2/20GROUP B MICE (20 grafts treated with the buffered saline solution) 02/20 4/20 7/20 1 8/20 6/20 3/20 2 6/20 5/20 5/20 3 4/20 5/20 5/20

[0071] The composition 1 improves the taking of the grafts of culturedhuman epidermis on athymic mice compared to a traditional survivalsolution (PBS). Significant differences are noted from 7 days oftreatment onwards, for a final improvement of more than 50%.

[0072] b) The Cicatrization (with the Grafted Whole Skins)

[0073] Scoring is performed from 0 to 3: 0=no cicatrization disorder;1=delay of cicatrization; 2=delay with abnormality of the cicatrization(granulation of the cicatrix); 3=hypertrophic cicatrix.

[0074] The results are collated in Table 3. TABLE 3 Score D-7 D-15 D-30GROUP A MICE (20 grafted whole skins treated with the nutrientcomposition) 0 20/20  16/20  15/20  1 0/20 3/20 2/20 2 0/20 1/20 2/20 30/20 0/20 1/20 GROUP B MICE (20 grafted whole skins treated with thebuffered saline solution) 0 16/20  10/20  5/20 1 4/20 7/20 8/20 2 0/203/20 3/20 3 0/20 0/20 4/20

[0075] The composition 1 significantly improves the cicatrizationprocesses; this effect is especially marked after 30 days of treatment.

1-22. (Canceled)
 23. A method of cosmetic treatment, comprisingcontacting only an area of human skin whose integrity has not beenbreached by injury with a treatment composition comprising an aqueouscomplex nutritive base comprising: a plurality of amino acids, at leastone vitamin, a plurality of assimilable organic components each selectedfrom the group consisting of i-Inositol, Putrescine 2HCl, Sodiumpyruvate, Thymidine, Adenine (HCl), DL-Lipoic acid and D-Glucose, and atleast one inorganic salt, wherein said treatment composition does notcomprise a biological extract of animal or cellular origin, or a livingnourishing substrate, or a cellular growth stimulating compound orfactor, or a hormone.
 24. The method of claim 23, wherein the pH andosmolarity of said complex nutritive base are per se close tophysiological conditions.
 25. The method of claim 23, wherein saidtreatment composition consists essentially of components that arebiomimetic to skin.
 26. The method of claim 23, wherein said amino acidsinclude at least one amino acid selected from the group consisting ofL-Alanine, L-Arginine HCl, L-Asparagine, L-Aspartic acid, L-CysteineHCl.H₂O, L-Glutamic acid, L-Glutamine, Glycine, L-Histidine HCl.H₂O,L-Isoleucine, L-Leucine, L-Lysine HCl, L-Methionine, L-Phenylalanine,L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2 Na 2H₂O,and L-Valine.
 27. The method of claim 23, wherein said at least onevitamin includes at least one vitamin selected from the group consistingof d-Biotin, Folic acid, Nicotinamide, Ca D-Pantothenate, PyridoxineHCl, Riboflavin, Thiamine HCl, and Vitamin B₁₂.
 28. The method of claim23, wherein said complex nutritive base supports per se viable in vitrogrowth of human epidermal keratinocytes, with no proliferation of anytransformed human epidermal keratinocyte.
 29. The method of claim 23,wherein the complex nutritive base does not have per se cytotoxicmanifestations to skin.
 30. The method of claim 23, wherein said methodis for maintaining the integrity and balance of the superficial cells ofthe skin.
 31. A method of cosmetic treatment, comprising contacting onlyan area of human skin whose integrity has not been breached by a woundwith a treatment composition comprising an aqueous complex nutritivebase comprising: a plurality of amino acids, at least one vitamin, aplurality of assimilable organic components each selected from the groupconsisting of i-Inositol, Putrescine 2HCl, Sodium pyruvate, Thymidine,Adenine (HCl), DL-Lipoic acid and D-Glucose, and at least one inorganicsalt, wherein said treatment composition does not comprise a biologicalextract of animal or cellular origin, or a living nourishing substrate,or a cellular growth stimulating compound or factor, or a hormone. 32.The method of claim 31, wherein the pH and osmolarity of said complexnutritive base are per se close to physiological conditions.
 33. Themethod of claim 31, wherein said treatment composition consistsessentially of components that are biomimetic to skin.
 34. The method ofclaim 31, wherein said amino acids include at least one amino acidselected from the group consisting of L-Alanine, L-Arginine HCl,L-Asparagine, L-Aspartic acid, L-Cysteine HCl.H₂O, L-Glutamic acid,L-Glutamine, Glycine, L-Histidine HCl.H₂O, L-Isoleucine, L-Leucine,L-Lysine HCl, L-Methionine, L-Phenylalanine, L-Proline, L-Serine,L-Threonine, L-Tryptophan, L-Tyrosine 2 Na 2H₂O, and L-Valine.
 35. Themethod of claim 31, wherein said at least one vitamin includes at leastone vitamin selected from the group consisting of d-Biotin, Folic acid,Nicotinamide, Ca D-Pantothenate, Pyridoxine HCl, Riboflavin, ThiamineHCl, and Vitamin B₁₂.
 36. The method of claim 31, wherein said complexnutritive base supports per se viable in vitro growth of human epidermalkeratinocytes, with no proliferation of any transformed human epidermalkeratinocyte.
 37. The method of claim 31, wherein the complex nutritivebase does not have per se cytotoxic manifestations to skin.
 38. Themethod of claim 31, wherein said method is for maintaining the integrityand balance of the superficial cells of the skin.
 39. A method ofcosmetic treatment, comprising contacting human skin with a treatmentcomposition comprising an aqueous complex nutritive base comprising: aplurality of amino acids, at least one vitamin, a plurality ofassimilable organic components each selected from the group consistingof i-Inositol, Putrescine 2HCl, Sodium pyruvate, Thymidine, Adenine(HCl), DL-Lipoic acid and D-Glucose, and at least one inorganic salt,wherein said treatment composition does not contain a biological extractof animal or cellular origin, or a living nourishing substrate, andwherein said treatment composition consists essentially of componentsthat are biomimetic to skin.
 40. The method of claim 39, wherein saidcomplex nutritive base supports per se viable in vitro growth of humanepidermal keratinocytes.
 41. The method of claim 39, wherein the pH andosmolarity of said complex nutritive base are per se close tophysiological conditions.
 42. The method of claim 39, wherein said aminoacids include at least one amino acid selected from the group consistingof L-Alanine, L-Arginine HCl, L-Asparagine, L-Aspartic acid, L-CysteineHCl.H₂O, L-Glutamic acid, L-Glutamine, Glycine, L-Histidine HCl.H₂O,L-Isoleucine, L-Leucine, L-Lysine HCl, L-Methionine, L-Phenylalanine,L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine 2 Na 2H₂O,and L-Valine.
 43. The method of claim 39, wherein said at least onevitamin includes at least one vitamin selected from the group consistingof d-Biotin, Folic acid, Nicotinamide, Ca D-Pantothenate, PyridoxineHCl, Riboflavin, Thiamine HCl, and Vitamin B₁₂.
 44. The method of claim39, wherein the complex nutritive base does not have per se cytotoxicmanifestations to skin.
 45. The method of claim 39, wherein said methodis for maintaining the integrity and balance of the superficial cells ofthe skin.
 46. A cosmetic composition, comprising an aqueous complexnutritive base comprising: a plurality of amino acids, at least onevitamin, a plurality of assimilable organic components each selectedfrom the group consisting of i-Inositol, Putrescine 2HCl, Sodiumpyruvate, Thymidine, Adenine (HCl), DL-Lipoic acid and D-Glucose, and atleast one inorganic salt, wherein said cosmetic composition does notcomprise a biological extract of animal or cellular origin, or a livingnourishing substrate, or a cellular growth stimulating compound orfactor, or a hormone.
 47. The cosmetic composition of claim 46, whereinthe pH and osmolarity of said complex nutritive base are per se close tophysiological conditions.
 48. The cosmetic composition of claim 46,wherein said cosmetic composition consists essentially of componentsthat are biomimetic to skin.
 49. The cosmetic composition of claim 46,wherein said amino acids include at least one amino acid selected fromthe group consisting of L-Alanine, L-Arginine HCl, L-Asparagine,L-Aspartic acid, L-Cysteine HCl.H₂O, L-Glutamic acid, L-Glutamine,Glycine, L-Histidine HCl.H₂O, L-Isoleucine, L-Leucine, L-Lysine HCl,L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine,L-Tryptophan, L-Tyrosine 2 Na 2H₂O, and L-Valine.
 50. The cosmeticcomposition of claim 46, wherein said at least one vitamin includes atleast one vitamin selected from the group consisting of d-Biotin, Folicacid, Nicotinamide, Ca D-Pantothenate, Pyridoxine HCl, Riboflavin,Thiamine HCl, and Vitamin B₁₂.
 51. The cosmetic composition of claim 46,wherein said complex nutritive base supports per se viable in vitrogrowth of human epidermal keratinocytes, with no proliferation of anytransformed human epidermal keratinocyte.
 52. The cosmetic compositionof claim 46, wherein the complex nutritive base does not have per secytotoxic manifestations to skin.
 53. A cosmetic composition, comprisingan aqueous complex nutritive base comprising: a plurality of aminoacids, at least one vitamin, a plurality of assimilable organiccomponents each selected from the group consisting of i-Inositol,Putrescine 2HCl, Sodium pyruvate, Thymidine, Adenine (HCl), DL-Lipoicacid and D-Glucose, and at least one inorganic salt, wherein saidcosmetic composition does not contain a biological extract of animal orcellular origin, or a living nourishing substrate, and wherein saidcosmetic composition consists essentially of components that arebiomimetic to skin.
 54. The cosmetic composition of claim 53, whereinsaid complex nutritive base supports per se viable in vitro growth ofhuman epidermal keratinocytes.
 55. The cosmetic composition of claim 53,wherein the pH and osmolarity of said complex nutritive base are per seclose to physiological conditions.
 56. The cosmetic composition of claim53, wherein said amino acids include at least one amino acid selectedfrom the group consisting of L-Alanine, L-Arginine HCl, L-Asparagine,L-Aspartic acid, L-Cysteine HCl.H₂O, L-Glutamic acid, L-Glutamine,Glycine, L-Histidine HCl.H₂O, L-Isoleucine, L-Leucine, L-Lysine HCl,L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine,L-Tryptophan, L-Tyrosine 2 Na 2H₂O, and L-Valine.
 57. The cosmeticcomposition of claim 53, wherein said at least one vitamin includes atleast one vitamin selected from the group consisting of d-Biotin, Folicacid, Nicotinamide, Ca D-Pantothenate, Pyridoxine HCl, Riboflavin,Thiamine HCl, and Vitamin B₁₂.
 58. The cosmetic composition of claim 53,wherein the complex nutritive base does not have per se cytotoxicmanifestations to skin.
 59. The cosmetic composition of claim 53, saidaqueous complex nutritive medium comprising at least four of saidassimilable organic components.